title: Spatially isotropic four-dimensional imaging with dual-view plane illumination microscopy.
abstract: Optimal four-dimensional imaging requires high spatial resolution in all dimensions, high speed and minimal photobleaching and damage. We developed a dual-view, plane illumination microscope with improved spatiotemporal resolution by switching illumination and detection between two perpendicular objectives in an alternating duty cycle. Computationally fusing the resulting volumetric views provides an isotropic resolution of 330 nm. As the sample is stationary and only two views are required, we achieve an imaging speed of 200 images/s (i.e., 0.5 s for a 50-plane volume). Unlike spinning-disk confocal or Bessel beam methods, which illuminate the sample outside the focal plane, we maintain high spatiotemporal resolution over hundreds of volumes with negligible photobleaching. To illustrate the ability of our method to study biological systems that require high-speed volumetric visualization and/or low photobleaching, we describe microtubule tracking in live cells, nuclear imaging over 14 h during nematode embryogenesis and imaging of neural wiring during Caenorhabditis elegans brain development over 5 h.
authors.affiliation: Section on High Resolution Optical Imaging, National Institute of Biomedical Imaging and Bioengineering, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: Yicong
authors.initials: Y
authors.lastname: Wu
authors.forename: Peter
authors.initials: P
authors.lastname: Wawrzusin
authors.forename: Justin
authors.initials: J
authors.lastname: Senseney
authors.forename: Robert S
authors.initials: RS
authors.lastname: Fischer
authors.forename: Ryan
authors.initials: R
authors.lastname: Christensen
authors.forename: Anthony
authors.initials: A
authors.lastname: Santella
authors.forename: Andrew G
authors.initials: AG
authors.lastname: York
authors.forename: Peter W
authors.initials: PW
authors.lastname: Winter
authors.forename: Clare M
authors.initials: CM
authors.lastname: Waterman
authors.forename: Zhirong
authors.initials: Z
authors.lastname: Bao
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Colón-Ramos
authors.forename: Matthew
authors.initials: M
authors.lastname: McAuliffe
authors.forename: Hari
authors.initials: H
authors.lastname: Shroff
mesh_terms: D000818:Animals; D001921:Brain / Q000033:anatomy & histology / Q000254:growth & development / Q000648:ultrastructure; D017173:Caenorhabditis elegans / Q000196:embryology; D058948:Cell Tracking / Q000379:methods*; D061307:Human Umbilical Vein Endothelial Cells; D006801:Humans; D021621:Imaging, Three-Dimensional / Q000379:methods*; D008029:Lighting; D008853:Microscopy / Q000295:instrumentation* / Q000379:methods*; D038761:Photobleaching